This invention relates to compositions, methods and kits for determining the presence and/or amount of each of two or more isomeric analytes in a sample suspected of containing the isomeric analytes.
Many small molecule compounds or haptens such as, for example, drugs and vitamins, exist in isomeric forms, of which only one form is active. In order to obtain an accurate measurement of the active form of an analyte, the presence of the non-active isomer of the analyte must be addressed. Measurements of both isomeric forms of an analyte, that is, active and non-active forms, can lead to inaccuracies that may be detrimental to an individual depending on the function of the active form of the analyte. Accurately assessing the level of each of a pair of isomeric analytes in biological samples is important especially where only one of the isomers is active and measurements that include the amount of the non-active isomer distort the level of the analyte in a sample. For example, measuring vitamin D levels in biological samples is important since vitamin D deficiency is related to a number of disorders in mammals. In infants, for example, vitamin D measurements that include amounts of 3-epi isomers can lead to inaccurate assessment of vitamin D levels in the infant, which in turn can lead to a lack of proper supplementation. It is important to measure the active form of vitamin D so that an infant can receive proper vitamin D therapy, if necessary.
The term “vitamin D” refers to a group of fat-soluble secosteroids. In humans, vitamin D is unique because it can be ingested as cholecalciferol (vitamin D3) or ergocalciferol (vitamin D2) and because the body can also synthesize it (from cholesterol) when sun exposure is adequate. Because of this latter property, vitamin D is considered by some to be a non-essential dietary vitamin although most consider it an essential nutrient. Vitamin D has an important physiological role in the positive regulation of calcium ion homeostasis. Vitamin D3 is the form of the vitamin synthesized by animals. It is also a common supplement added to milk products and certain food products as is vitamin D2.
Both dietary and intrinsically synthesized vitamin D3 must undergo metabolic activation to generate bioactive metabolites. In humans, the initial step of vitamin D3 activation occurs primarily in the liver and involves hydroxylation to form the intermediate metabolite 25-hydroxycholecalciferol (also referred to as calcidiol, calcifediol, 25-hydroxycholecalciferol, or 25-hydroxyvitamin D3. Calcidiol is the major form of Vitamin D3 in the circulatory system. Vitamin D2 also undergoes similar metabolic activation to 25-hydroxyvitamin D2. Collectively these compounds are called 25-hydroxyvitamin D (abbreviated 25(OH)D) and they are the major metabolites that are measured in serum to determine vitamin D status; 25(OH)D and its epimers are both pre-hormones that need to be converted into 1,25(OH)D to exert biological functions. The comparison of bioactivity of 1,25(OH)D versus that of 3-epi-1,25(OH)D is complex.
The vitamin D compounds 25-hydroxyvitamin D3 and 25-hydroxyvitamin D2 are epimeric at the 3-position with the epimers being designated 25-hydroxyvitamin D3 and 3-epi-25-hydroxyvitamin D3 and 25-hydroxyvitamin D2 and 3-epi-25-hydroxyvitamin D2, respectively. Only one of the epimers of each of these epimeric compounds, namely, 25-hydroxyvitamin D3 and 25-hydroxyvitamin D2, respectively, are active. The structures for the epimers of 25-hydroxyvitamin D3 and 25-hydroxyvitamin D2 are set forth in FIG. 1.
There is a need for reagents and methods for accurate and sensitive determinations of concentrations of isomeric analytes in samples suspected of containing such analytes. For example, there is a need for reagents and methods for accurate and sensitive determinations of concentrations of epimeric forms of vitamin D.